KR101245154B1 - Vaccine composition against hepatitis c virus - Google Patents

Abstract

The present invention is a vaccine composition for the treatment and prophylactic treatment of hepatitis C, which comprises the hepatitis C virus structural antigen as a component in a proportion and has a proton effect in the development of an immune response against hepatitis C virus. Combined vaccines against pathogens comprising this vaccine composition are also described.

Hepatitis C virus, vaccine, structural protein E1, N. meningitidis

Description

Vaccine composition for hepatitis C virus {VACCINE COMPOSITION AGAINST HEPATITIS C VIRUS}

TECHNICAL FIELD The present invention relates to the field of immunology, and in particular, to vaccine antigen compositions capable of eliciting a potent and diverse response to hepatitis C virus (HCV) and combined vaccines against pathogens comprising the vaccine composition.

As RNA viruses, some of these disorders have hindered the production of effective vaccines against HCV because they can be rapidly mutated in their adaptation to the environment. This contributes to a wide variety of sequences from complex virus isolates identified around the world. The major heterogeneity is concentrated in highly diversified regions, specifically neutralizing epitopes, framed in the HCV E2 protein ( Bukh meat al . US 6,110,465). HCV causes persistent infections in individuals with immunity despite the presence of an active immune response (Lechmann et al. (2000) Vaccine development for hepatitis C. Semin Liver Dis . 20: 211-226). There is no ex vivo cell culture system or effective animal model that supports the assessment of the presence of neutralizing antibodies and HCV replication. The immunological patterns associated with disease progression or protection are not fully defined. Strong, multispecific, long-term, humoral and cellular immune responses will be required to elucidate HCV infection (Lechmann et al. (2000) Vaccine development for hepatitis C. Semin Liver Dis . 20: 211-226).

Several approaches have been used to develop vaccines for HCV; Among them, viruses such as recombinant proteins, synthetic peptides, particles, bare DNA and recombinant viruses have been widely evaluated ( Depla meat al . US 6,635,257; Liang et al. US 6,387,662; Pachuk meat al . US 6,235,888; Berzofsky meat al . US 6,685,944).

Since antibodies against envelope proteins confer protection against some flaviviruses, the development of vaccines based on protein subunits is one of the strategies already evaluated for HCV ( Min meat al . US 5,985,609). Several strategies based on HCV structural antigens have led to limited protection against viruses in animal models. This is the case for chimpanzees immunized with oligomers of El and E2. Seven chimpanzees were vaccinated, five of the seven were protected and two were ill but treated before reaching a chronic condition (Choo et al. (1994) Vaccination of chimpanzees against infection by hepatitis C virus. PNAS USA , 91: 1294-98). This protection is related to the presence of antibodies (Abs) that can inhibit the binding of E2 to human cells (Rosa et al. (1996) A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: Cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.PNAS USA , 93: 1759-63).

From the genotype 1b isolate, recombinant E1 protein was purified with homodimers associated with particles of approximately 9 nm size (Maertens et al. (2000) Improvement of chronic active hepatitis C in chronically infected chimpanzees after therapeutic vaccination with the HCV E1 protein Acta Gastroenterol Belg . 63: 203). Two chimpanzees were chronically infected with 50 μg recombinant E1 protein at 9 doses of HCV uptake. This vaccination improved liver tissue structure and determined the disappearance of viral antigens in the liver and the reduction of alanine aminotransferase levels (ALT). Although the level of ARN in serum did not change during treatment, hepatic inflammation and viral antigens reappeared after treatment termination. The correlation between the improvement of the high levels of antibodies to the E1 and disease was observed (Maertens et al. (2000) Improvement of chronic active hepatitis C in chronically infected chimpanzees after therapeutic vaccination with the HCV E1 protein. Acta Gastroenterol . Belg . 63: 203).

The formation of viruses such as particles from recombinant proteins and their use as vaccines is very attractive because their structure is very similar to the characteristics of the virus ( Liang meat al . US 6,387,662) . These native particles from insect cells infected with recombinant baculovirus carrying a sequence of HCV structural antigens can elicit a humoral and cellular immune response to these antigens (Baumert et al. (1999) Hepatitis C virus— . like particles synthesized in insect cells as a potential vaccine candidate Gastroenterology 117:. 1397-407; Lechmann et al (1999) Induction of humoral and cellular immune responses in mice by immunization with insect-cell derived HCV-like particles Hepatology 30:. 423-29).

Meanwhile, several viral recombinant vectors have been evaluated to develop recombinant vaccines against HCV. In particular, recombinant disordered adenoviruses are attractive candidates for their ability to be administered by parenteral and oral routes, as well as their hepatitis, which is the ability to induce immunity of body fluids and cells. The recombinant adenovirus containing the gene encoding the HCV structural proteins induce an antibody response against those proteins (Makimura et al. (1996) . Induction of antibodies against structural proteins of hepatitis C virus in mice using recombinant adenovirus. Vaccine 14 : 28-36). In addition, after mouse immunization with recombinant adenovirus comprising the core and E1 antigens, T cytotoxic specific responses to these antigens were detected (Bruna-Romero et al. (1997) Induction of cytotoxic T-cell response against hepatitis C virus structural antigens using a defective recombinant adenovirus Hepatology 25:. 470-77). Although these responses are encouraging, recent problems with the use of recombinant adenoviruses in gene therapy have raised some questions about their use in the human body. Adoption of other recombinant viruses, such as vaccinia virus, which carries other HCV genes, induced strong T cytotoxicity and helper responses in mice (Shirai et al. (1994) An epitope in hepatitis C virus core region recognized by cytotoxic T cells in . mice and humans J. Virol 68: .... 3334-42; Large et al (1999) Suppression of host immune response by the core protein of hepatitis C virus: possible implications for hepatitis C virus persistence J. Immunol 162: 931 -38). Nevertheless, the use of these recombinant viruses as well as other variants of alpha viruses, such as the semliki forest virus, are affected by the controlled and safe issues associated with their application (Vidalin et al. (2000) Use). of conventional or replicating nucleic acid-based vaccines and recombinant Semliki forest virus-derived particles for the induction of immune responses against hepatitis C virus core and E2 antigens Vaccine 276:. 259-270).

The identification of several T CD4 + and CD8 + epitopes in viral HCV polyproteins, which may be important for the classification of viruses, supports the strategy of using synthetic peptides as vaccine candidates for this pathogen (Berzofsky et al. US 5,980,899). Other peptides, alone or lipidated, containing HCV epitopes from the core, NS4 and NS5 proteins induced strong T cytotoxic responses in mice (Shirai et al. (1996) Use of intrinsic and extrinsic helper epitopes for in vivo induction of anti-hepatitis C virus cytotoxic T lymphocytes (CTL) with CTL epitope peptide vaccines J. Infect Dis 173:.... 24-31; Hiranuma et al (1999) Helper T cell determinant peptide contributes to induction of cellular immune responses by peptide ... vaccines against hepatitis C virus J. Gen Virol 80: 187-193; Oseroff et al (1998) Pools of lipidated HTL-CTL constructs prime for multiple HBV and HCV CTL epitope responses Vaccine, 16:.. 823-833) .

Another strategy used to develop vaccines against HCV is due to the possibility of generating antibodies against linear epitopes. This alternative has been primarily evaluated for generating antibodies against HCV hypervariable region 1 (HVR-1) in rabbits and chimps with some encouraging results (Esumi et al. (1999) Experimental vaccine activities of recombinant E1 and E2 glycoproteins and hypervariable region 1 peptides of hepatitis C virus in chimpanzees. Arch Virol . 144: 973-980; Shang et al. (1999) Broadly cross-reactive, high-affinity antibody to hypervariable region 1 of the hepatitis C virus in rabbits. Virology 258: 396-405). The main problem in selecting HVRs as a target for HCV vaccines is due to the presence of the quasi-species in this genome region.

The major obstacle to the peptide vaccine approach is that these peptides without helper action may be poor immunogens, and the efficacy of the vaccine is mainly based on the induction of multiple responses to a very broad range for different antigens. Their limitations are a disadvantage of this strategy.

DNA immunization has been extensively studied for vaccine development against HCV (Donnelly et. al . US 6,653,125). The core protein is expressed in several expression vectors, the full length or truncated of this protein. It was included by using the elastic element (Lagging et al (1995) Immune responses to plasmid DNA encoding the hepatitis C virus core protein J. Virol 69:.... 5859-5863; Chen et al (1995) Genetic immunization of mice with plasmid containing hepatitis C virus core protein-encoding DNA.Vaccine Res . 4: 135-144).

Other genetic constructs also include 5 non-transcriptional regions (Tokushige et al. (1996) Expression and immune response to hepatitis C virus core DNA-based vaccine constructs. Hepatology 24: 14-20). Variants fused to HB surface antigens and variants from other viruses have also been studied (Major et al. (1995) DNA-based immunization with chimeric vectors for the induction of the immune responses against the hepatitis C virus nucleocapsid. J. Virol . 69: 5798-805). Immunization reactions with these vectors induced mainly detectable lymphoproliferative and T cell lymphocyte cytotoxic responses.

HCV envelope proteins also constitute interesting targets for this kind of technology. In the case of E2, the humoral immune response is believed to be for HVR-1 (Lee et al. (1998) Hepatitis C virus envelope DNA-based immunization elicits humoral and cellular immune responses. Mol . Cells 8: 444-451). When the immunization reaction was performed with vectors for secretable variants of E1 and E2 proteins, no differences in immune responses were detected compared to non-secretable variants (Lee et al. (1998) Optimal induction of hepatitis C virus envelope -specific immunity by bicictronic plasmid DNA inoculation with the granulocyte-macrophage colony-stimulating factor gene J. Virol 72:.. 8430-8436). Inoculation with bicistronic plasmids expressing independently the genes codifying for the GM-CSF, inoculation with non-cistronic plasmids that independently express genes encoding the proteins developed and enhanced humoral and cellular immune responses. Recently, the immunological effects of these proteins have been investigated where the possibility of heterodimer formation in vivo is supported or eliminated when both are included in non-cistronic vectors. When these complexes were formed, it was confirmed that no antibody reaction was obtained. In a close comparison, high antibody titers for conformational and linear determinants occurred when the animal was immunized with a plasmid expressing a turnedcated form of quantum structural protein. Therefore, it is considered necessary to avoid heterodimer formation in order to obtain an excellent antibody response when immunization with both antigens is performed (Fournillier et al. (1999) Expression of noncovalent hepatitis C virus envelope E1-E2 complexes is not required . for the induction of antibodies with neutralizing properties following DNA immunization J. Virol 73:. 497-504).

Nonstructural proteins were also evaluated through this technique. The region encoding for the C-terminal region of the NS3 protein has been included in a vector that allows simultaneous or independent expression of this protein and IL-2 with good results (Papa et al. (1998) .. multigenetic plasmid vector for HCV DNA immunization Res Virol 149:. 315-319). NS4 and NS5 proteins are the lymphocytes T cytotoxic and antibody responses by the same method were generated (Encke et al. (1998) Genetic immunization generates cellular and humoral immune responses against the nonstructural proteins of the hepatitis C virus in murine model. J. Immunol . 161: 4917-4923).

Recently, the use of gene constructs that encode viral nonstructural proteins (NS3, NS4 and NS5; including genes encoding for GM-CSF) has increased the T cell proliferative response to each one of the nonstructural proteins. it was confirmed that not only that a strong antibody response generated (Cho et al (1999) Enhanced cellular immunity to hepatitis C virus nonstructural proteins by codelivery of granulocyte macrophage-colony stimulating factor gene in intramuscular DNA immunization Vaccine 17:.. 1136-1144) .

In general, occurrence as well as effective expression of antibodies to other HCV antigens after DNA immunization reactions have been reported. The level in the research is the area between 100,000: 1 depending on the combination of the (Inchauspe et al (1997) DNA vaccination for the induction of immune responses against hepatitis C virus proteins Vaccine 15.. 853-856): 100 to 1 . In addition, the specific cytotoxicity and lymphocyte proliferation have been identified improvement of (Inchauspe et al. (1997) Plasmid DNA expressing a secreted or a nonsecreted form of hepatitis comparative studies of antibody and T-helper responses following genetic immunization. DNA and Cell Biology 16: 185-195). However, it is necessary to improve this methodology in order to arrive at a sufficiently strong humoral and cellular immune response against other HCV proteins. In this regard, several variants have been evaluated to improve the immune response induced after DNA vaccination, which has been described as Gramzinski et al. (1998) Immune response to a hepatitis B DNA vaccine in Aotus Monkey : A comparison of vaccine formulation, route and method of administration Mol Medicine 4:.. 109-118), monophosphoryl lipid A and the saponin QS-21 (Sasaki et al ( 1998.) Induction of systemic and mucosal immune responses to human immunodeficieny virus type . 1 by a DNA vaccine formulated with QS-21 saponin adjuvant via intramuscular and intranasal routes J. Virol 72:. the use of 4931-4939). On the other hand, dentit cells were studied as biological adjuvant in DNA immunization reaction. Vaccines consisting of dentit cells derived from genetically modified mouse bone marrow in vitro were used to express tumor antigens using viral vectors, and specific T cell responses and prophylactic immunity mediated by cells to tumors in mice. His ability to stimulate narcosis has been demonstrated (Specht et al. (1997) Dendritic cells retrovirally transduced with a model antigen gene are therapeutically effective against established pulmonary metastases. J. Exp . Med . 186: 1213-1221; Brossart et al. (1997) Virus-mediated delivery of antigenic epitopes into dendritic cells as a means to induce CTL. J. Immunol . 158: 3270-3276; Song et al. (1997) Dendritic cells genetically modified with an adenovirus vector encoding the cDNA for a .. model antigen induce protective and therapeutic antitumor immunity J. Exp Med 186:. 1247-1256), o ARN (. Boczkowski et al (1996) Dendritic cells pulsed with RNA are potent antigen-presenti ng cells in in vitro and in vivo . J. Exp . Med . 184: 465-472).

Currently, improvements in vectors involving the insertion of CpG have been carried out to boost the immune response to expressed antigens, and systems for plasmid release are critical targets in studies to overcome the limitations of this technique (Hasan et al. . (1999) Nucleic acid inmunization: . concepts and techniques associated with third generation vaccines J. Immunol Meth 229:.. 1-22).

Due to the lack of a clear definition of the pathogen introduction established by HCV and the immunological parameters associated with protection against this pathogen, an effective vaccine against HCV may require a variable approach that stimulates various aspects of the immune response. have. The solution to this problem lies in the combination of several vaccine approaches tested to date. In this regard, the more positive immunization schedule, which combines an initial dose with a DNA vaccine and an additional dose with a protein or recombinant viral vector, is an additional investigation to demonstrate that the combination of subjects can induce protective immunity against HCV. It has to be evaluated as a result it requires (Hu et al (1999) Characterization of the humoral and cellular immune responses against hepatitis C virus core induced by DNA-based immunization Vaccine 17:... 3160-3170; Pancholi et al (2000 ) DNA prime-canarypox boost with polycistronic hepatitis C virus (HCV) genes generates potent immune responses to HCV structural and nonstructural proteins J. Infect Dis 182:... 18-27).

In addition, for the hepatitis B model, a vaccine formulation consisting of a complex of hepatitis B surface antibody with a monoclonal antibody against HbsAg and a plasmid encoding for this antigen was evaluated (Wen et al. US 6,221,664). This formulation allows for the rapid induction of a higher immune response than that generated by the subject separated from the expression of the antigen by other pathways. In this way, the development of effective vaccines is a great and even more significant success, which is still an ongoing task.

In the present invention, vaccine formulations composed of structural antigens of hepatitis C virus are described. Compared to conventional vaccine formulations based on proteins, when only E1 and E2 proteins are used (alone or in combination), the vaccine formulation also includes HCV core antigens. The flexibility of this vaccine composition is in the category of antigen amounts used in this vaccine formulation, which allows a strong immune response to other antigens in a simultaneous manner as well as a positive response to pathogen introduction into the recombinant vaccinia virus in the murine model. And cellular) development additionally.

It is established in the literature that it is urgent to develop reliable and effective therapeutic vaccines for HCV. Accordingly, the present invention has focused on the supply of vaccine formulations composed of a mixture of structural antigens of hepatitis C virus. In this case, the core protein, structural protein E1 and structural protein E2 can induce humoral and cellular specific immune responses against HCV, even for chronic patients of HCV.

The novelty of the present invention was given by obtaining a range of protective effects and antigen correlations obtained after immunization reactions with mixtures in this study. Antigens in the study are attractive vaccine targets by means that immune responses can be generated for HCV.

In certain embodiments, the vaccine formulation is tailored to (1.5-2.5) :( 1-2.5) :( 1-2) and present in a mixture for each of the core proteins, E1 and E2, to generate an optimal humoral immune response. Use a range of correlations for the antigen. In another embodiment, antigens were used with a correlation of (1.5: 1.1: 1) for the core proteins, El and E2, respectively.

In an embodiment of the invention, the vaccine formulation is in a mixture adjusted to (1:50) :( 120-180) :( 120-180) for each of the core proteins, E1 and E2, in order to generate an optimal cellular immune response. Use a range of correlations for the antigen present. In another embodiment, protein antigens were used in correlation of (1: 160: 160) for the core proteins, El and E2, respectively.

Vaccine formulations of the present invention may be administered as a solid product, liquid product or aerosol by convenient methods for vaccine administration, including oral and parenteral injection (ie, intravenously, subcutaneously or intramuscularly), and furthermore the composition Silver may be prepared by admixing the protein antigens prior to adjuvant as well as by adjuvant the antigens separately and later mixing them.

Antigens according to the present invention can be obtained in nature or by recombinant DNA techniques in any expression system such as provided below. In an embodiment of the present invention, the vaccine formulation included in the present invention may be administered on different immunization schedules.

In the same way, immunization with these antigenic combinations mixed with antigens of other polysaccharides (conjugated or not) and / or viral and / or bacterial antigens thus generates a synthetic immunization response against a wide range of infectious pathogens.

In another embodiment of the invention, the vaccine formulation comprises a combination of HCV structural antigens with plasmids encoding for antigens from infectious pathogens for viral and / or bacterial diseases.

Others are preferred vaccine composition in the present invention including viral antigen: from the hepatitis B core and / or surface antigens, TB foil O antigen as N. mening proteosome liposomes or N. mening giti disk (N. meningitides) from the discharge giti And polysaccharide antigens that are conjugated to or without purified outer membrane proteins and carrier proteins. They can be used as active subjects in formulations for viral and bacterial pathogens.

The vaccine compositions described herein can be administered on a schedule combined with other vaccine candidates based on live recombinant vectors, peptides and proteins that are DNA vaccines. It can also be used to induce immunity to HCV in chronic hepatitis C patients with cirrhosis and liver cancer.

1 is a humoral immune response to HCV structural proteins assessed by ELISA, (A) for HCV core antigen, (B) for E1 protein, (C) for E2 protein.

2 is a lymphoproliferative immune response to HCV structural proteins in the spleen cytoplasm from mice immunized in combination in the study. HCcAg, core antigen of HCV, E2-coli, E2 produced and purified from E. coli, E2, E2-lev, yeast Pichia pastoris and purified E2, E1-coli, E1 produced and purified from E. coli.

3 is the functional response of the formulations in the study evaluated after introduction of the pathogen into the recombinant vaccinia virus.

4A, 4B and 4C are analyzes of the effects of composition on pathogen introduction into (A) humoral immune response, (B) lymphoproliferative immune response, and (C) recombinant vaccinia virus in the presence of HCV vaccine formulation.

FIG. 5 shows the effect on humoral immune responses by the correlation range of antigens used in HCV vaccine formulations, (A) for the core antigen of HCV, (B) for the E1 protein, (C) the E2 protein It is about.

FIG. 6 is an effect on lymphoproliferative immune response to HCV structural proteins in splenic cells of mice immunized by the correlation range of antigens used in HCV vaccine preparations. HCcAg, core antigen of HCV, E2-coli, E2 produced and purified from E. coli, E2, E2-lev, yeast Pichia pastoris and purified E2, E1-coli, E1 produced and purified from E. coli.

7 is the effect on the functional response evaluated in mice for pathogen introduction into recombinant vaccinia by the extent of correlation of antigen used in HCV vaccine formulations.

8 is a humoral immune response to HCV structural proteins assessed by ELISA after immunization at different time intervals (different immunization schedule), (A) for HCV core antigen, (B) for E1 protein, (C) for the E2 protein.

9 is a lymphoproliferative immune response to HCV structural proteins in splenic cells of mice immunized at different time intervals (different immunization schedules). HCcAg, HCV core antigen, E2-coli, E2 produced and purified from E. coli, E2-lev, E2 produced and purified from yeast Pichia pastries, E1 produced and purified from E. coli.

10 is a functional response to pathogen introduction into recombinant vaccinia virus of mice immunized at different time intervals.

11 is a humoral immune response to HCV structural proteins assessed by ELISA of mice immunized with HCV vaccine preparation by different routes, (A) against HCV core antigen, (B) against E1 protein, (C) for the E2 protein.

12 is a lymphoproliferative immune response to HCV structural proteins in splenic cells of mice immunized with HCV vaccine formulations administered by different routes. HCcAg, HCV core antigen, E2-coli, E2 produced and purified from E. coli, E2-lev, E2 produced and purified from yeast Pichia pastries, E1 produced and purified from E. coli.

FIG. 13 is a functional immune response evaluated for pathogen introduction into recombinant vaccinia virus in mice immunized with HCV vaccine formulation administered by different routes.

14 is a humoral immune response to HCV structural proteins assessed by ELISA of mice immunized with HCV vaccine formulation using different adjuvant, (A) for HCV core antigen, (B) for El protein (C) for the E2 protein.

FIG. 15 is the effect of another adjuvant on lymphoid proliferative immune response to HCV structural proteins in spleen cells of immunized mice. HCcAg, HCV core antigen, E2-coli, E2 produced and purified from E. coli, E2, E2-lev, yeast Pichia pastries and purified E2, E1-coli, E1 produced and purified from E. coli.

FIG. 16 is the effect of another adjuvant on functional immune response to pathogen introduction into recombinant vaccinia virus in immunized mice.

17 is a humoral immune response assessed by ELISA following immunization with a mixture of HCV vaccine formulation and viral antigen, (A) for HCV core antigen, (B) for E1 protein, (C) E2 protein For, (D) HbsAg And (E) for HbcAg.

FIG. 18 is a lymphoproliferative immune response to HCV structural proteins (core, E1, E2), HBsAg and HBcAg in spleen cells of mice immunized with a combination of HCV vaccine preparation and viral antigen. HCcAg, HCV core antigen, E2-coli, E2 produced and purified from E. coli, E2-lev, E2 produced and purified from yeast Pichia pastries, E1 produced and purified from E. coli.

19 is a functional immune response to pathogen introduction into recombinant vaccinia virus in mice immunized with a combination of HCV vaccine preparation and viral antigen.

Figure 20 shows HCV vaccine preparations and bacterial antigens ( N. meningitidis Humoral immune response assessed by ELISA after immunization with a mixture of outer membrane proteins from (A) against HCV core antigen, (B) against E1 protein, (C) against E2 protein And (D) yen. For outer membrane proteins from Meningitidis .

Figure 21 shows HCV vaccine formulations and bacterial antigens (OMP, N. meningitidis Of ene from spleen cells of mice immunized with a combination of outer membrane proteins) . Lymphoproliferative immune responses against outer membrane proteins from Meningitidis and HCV structural proteins (core, E1, E2). HCcAg, HCV core antigen, E2-coli, E2 produced and purified from E. coli, E2-lev, E2 produced and purified from yeast Pichia pastries, E1 produced and purified from E. coli.

FIG. 22 is a functional immune response to pathogen introduction into recombinant vaccinia virus in mice immunized with a combination of HCV vaccine formulation and bacterial antigen (outer membrane protein from N. meningitidis ).

23 is a humoral immune response assessed by ELISA of mice immunized with a mixture of HCV vaccine formulation and conjugated capsular polysaccharide, (A) for HCV core antigen, (B) for E1 protein, (C) for E2 protein and (D) yen. For capsular polysaccharide of serogroup C from Meningitidis .

FIG. 24 is a lymphoproliferative immune response to HCV structural proteins (core, E1, E2) in splenic cells of mice immunized with a combination of HCV vaccine formulation and conjugated encapsulated polysaccharide. HCcAg, HCV core antigen, E2-coli, E2 produced and purified from E. coli, E2-lev, E2 produced and purified from yeast Pichia pastries, E1 produced and purified from E. coli.

25 is a functional immune response to pathogen introduction into recombinant vaccinia virus in mice immunized with a mixture of HCV vaccine formulation and conjugated encapsulated polysaccharide.

26 is a humoral immune response assessed by ELISA following immunization with a HCV vaccine formulation and a mixture of plasmids encoding HBsAg and HBcAg, (A) for HCV core antigen, (B) for E1 protein, (C) for E2 protein, (D) for HBsAg, and (E) for HBcAg.

FIG. 27 is a lymphoproliferative immune response to HCV structural proteins (core, E1, E2), HBsAg and HBcAg in splenic cells of mice immunized with a combination of HCV vaccine formulation and plasmid encoding HBsAg and HBcAg. HCcAg, HCV core antigen, E2-coli, E2 produced and purified from E. coli, E2-lev, E2 produced and purified from yeast Pichia pastries, E1 produced and purified from E. coli.

Figure 28 is a functional immune response to pathogen introduction into recombinant vaccinia virus of mice immunized with a combination of an HCV vaccine formulation and a plasmid encoding HBsAg and HBcAg antigen.

29 is a humoral immune response assessed by ELISA of mice immunized with a combination of addition dose and DNA with HCV vaccine formulation, (A) for HCV core antigen, (B) for E1 protein, (C ) For the E2 protein.

FIG. 30 is a lymphoproliferative immune response to HCV structural proteins (core, E1, E2) in splenic cells of mice immunized with a combination of additional dose and DNA with HCV vaccine formulation. HCcAg, HCV core antigen, E2-coli, E2 produced and purified from E. coli, E2-lev, E2 produced and purified from yeast Pichia pastries, E1 produced and purified from E. coli.

Figure 31 is a functional immune response to the introduction of pathogens into recombinant vaccinia virus in mice immunized with a combination of additional doses and DNA with HCV vaccine preparation.

32 is a humoral immune response to HCV structural proteins (core, E1, E2) in mice immunized with HCV vaccine formulations made by another method.

33 is a lymphoproliferative immune response to HCV structural proteins (core, E1, E2) in splenic cells of mice immunized with HCV vaccine formulations made by another method. HCcAg, HCV core antigen, E2-coli, E2 produced and purified from E. coli, E2-lev, E2 produced and purified from yeast Pichia pastries, E1 produced and purified from E. coli.

34 is a functional immune response to pathogen introduction into recombinant vaccinia virus in mice immunized with HCV vaccine preparations made by different methods.

Example  One: HCV  As a protein vaccine formulation Immunized  For Pathogen Introduction into Recombinant Vaccinia Virus in Mice Humoral Lymphatic proliferative and functional immune responses.

To determine the development of specific and functional immune responses to HCV following administration of the vaccine formulation, HCV core, E1 and E2 antigens were mixed in aluminum hydroxide and adjuvanted to peritoneal female BALB / c mice (10 groups). Was injected into the pathway. Immunization schedules include 3 inoculations at 0, 7, 14 days. 15 days after the last inoculation, humoral and cellular immune responses (lymphogenic responses) were studied as well as protection against pathogen introduction with recombinant vaccinia virus vvRE (including HCV structural proteins). The groups in this study are shown in Table 1.

Preparation of immunogens for use in this study group Immunogen Inoculation volume Route One 8.37 μg HCcAg +16.8 μgE1 +8.37 μg E2
100 μL / mouse

Adjuvant: Aluminum Hydroxide
(1:40) :( prot: AlOH ₃ ) Intraperitoneally 2 8.37 μg HCcAg +8.37 μg E1 +8.37 μg E2 3 13.34 μg HCcAg + 3.34 μg E1 +13.34 μg E2 4 8.37 μg HCcAg +8.37 μg E1 +16.8 μg E2 5 3.34 μg HCcAg + 3.34 μg E1 + 3.34 μg E2 6 8.37 μg HCcAg + 50 ng E1 + 8.37 μg E2 7 16.8 μg HCcAg + 8.37 μg E1 + 8.37 μg E2 8 3.34 μg HCcAg + 13.34 μg E1 + 3.34 μg E2 9 3.34 μg HCcAg + 3.34 μg E1 + 13.34 μg E2 10 8.37 μg HCcAg + 8.37 μg E1 + 50 ng E2 11 13.34 μg HCcAg + 13.34 μg E1 + 3.34 μg E2 12 50 ng HCcAg + 8.37 μg E1 + 8.37 μg E2 13 3.34 μg HCcAg + 13.34 μg E1 + 13.34 μg E2 14 13.34 μg HCcAg +3.34 μg E1 + 3.34 μg E2 15 13.34 μg HCcAg +13.34 μg E1 + 13.34 μg E2 16 8.37 μg HCcAg + 8.37 μg E1 + 8.37 μg E2 17 Adjuvant: Aluminum Hydroxide

Humoral immune responses to HCV structural proteins were determined by immuno-enzymatic analysis (ELISA). The "Kruskal-Wallis One Way Analysis of Variance" and post-test "Dunn's Method" were used to statistically analyze the results. Results with p <0.05 are considered to have statistically significant differences. Figure 1 shows that when it is administered in any correlation it can obtain specific humoral immune responses against three HCV structural antigens. Some combinations confer high antibody titers on HCV proteins (core, El, E2), which are statistically higher than those obtained in controls in which mice were immunized with independent proteins or adjuvant alone.

Lymphoproliferative responses to HCV structural antigens (core, E1, E2) (FIG. 2) showed characteristic behavior in each combination. These data are then used to calculate the optimal variant (antigen correlation) to form the best cellular response. This result is indicated by the stimulation index of splenic cells from immunized animals (calculated by ³ H-thymidine coalescence). The experimental group shown in FIG. 2 also corresponds to that shown in Table 1 with a negative control (group 17) corresponding to mice immunized with aluminum hydroxide only.

The functional immune response generated by the formulations of this study was assessed after introduction of the pathogen with vvRE. The pathogen was introduced into the animals 15 days after the end of the immunization schedule with 10 ⁶ pfu vvRE by ip route, and the presence of the virus in the mouse ovary was evaluated 5 days after virus inoculation. Figure 3 shows the results obtained, group 13 showed 2 Log-reduction in virus titer compared to the negative control. The experimental group shown in FIG. 3 corresponds to that shown in Table 1.

Perform surface response analysis generated after correlation of the variables used (antibody response, lymphoproliferative response and protection against pathogen introduction into vaccinia virus (FIGS. 4A, 4B, 4C) to obtain optimal response for each of the variables. The best antigen correlation for was calculated. The optimal antigen correlation used for the antibody response corresponds to (1.5: 1.1: 1) :( core: E1: E2), whereas the antigen correlation used for immunization to obtain an optimal cellular response is (1: 160: 160) :( core: E1: E2).

Example  2: having a different range of antigenic correlation HCV  Assessment of immune response in BALB / c mice after immunization with protein preparations.

To assess the range of antigens used in vaccine formulations to obtain a good immune response, BALB / c mice were immunized with i.p. under the same immune schedule as described in Example 1. The correlation of HCV antigens (core, E1 and E2) is shown in Table 2. The amount of antigen used in this study is the result of the surface response analysis from Example 1. For humoral immune responses, the studied range of correlations was: (1.5-2.5) :( 1-2.5) :( 1-2) :( core: E1: E2), whereas for the cellular immune response The studied range of relationships is: (1:50) :( 120-180) :( 120-180): (core: E1: E2).

Humoral immune response to HCV structural proteins is shown in FIG. 5. The correlations studied resulted in specific antibody responses to HCV antigens (core, El and E2). Despite the variation observed for each specific antigen, no statistical difference could be found in the humoral response generated by other variants (optimal for cellular response and optimal for humoral response). The experimental group coordinated in FIG. 5 corresponds to that shown in Table 2.

Lymphoproliferative responses were assessed by ³ H-thymidine coalescence in spleen cells of mice immunized after their stimulation. Specific lymphoproliferative responses to the antigens studied were developed in immunized mice (FIG. 6). The group coordinated in FIG. 6 corresponds to that shown in Table 2, and the other control group (group 17) corresponds to the non-immunized group. No statistically significant differences were observed among the stimulation indices observed for each of the variants studied (optimal variants for cellular responses and optimal variants for humoral responses, respectively).

Immunized mice were introduced into the pathogen with a vvRE of 10 ⁶ pfu by the ip pathway. The titer of the virus was assessed in the ovaries of immunized mice 5 days after virus inoculation. 7 shows the achieved protection level. Reduction of viral load was statistically significant in mice immunized with the mixture as compared to the negative control group in the group of animals immunized with the optimal variant primarily to achieve a cellular immune response (approximately a decrease in viral titer of 2.5 Log). ). The analyzed group shown in FIG. 7 corresponds to that shown in Table 2, and the additional negative control (group 17) corresponds to the non-immunized group.

Example  3: other immunizations On schedule  According HCV  By protein immunization BALB From the / c mouse Generated  Evaluation of the Immune Response.

To analyze the effect of time during immunization in the development of an immune response, female BALB / c mice were immunized by ip pathways at different time intervals with variants for the generation of an optimal cellular immune response as mentioned in Example 1 It became. Group 1 was immunized at 1, 1 and 2 weeks. Group 2 was immunized at 0, 2, and 4 weeks. Group 3 was immunized at 0, 3, and 6 weeks, while group 4 was immunized at 0, 4, and 8 weeks. Antibody titers against HCV core, El and E2 proteins were assessed by ELISA. The result is shown in FIG. There were no statistically significant differences in antibody titers against E1 and E2 when they were assessed 15 days after the last immunization. Significant statistical differences were observed in antibody titers against the core antigen when they were compared between group 1 (immunization schedule at weeks 0, 1 and 2) and the rest of the group.

Lymphoproliferative responses in immunized mice were evaluated 15 days after the last immunization. Like the difference in stimulus index among each studied variant, they do not have statistically significant differences. The results obtained are shown in FIG. 9, where additional negative controls are also included (group 5) and correspond to non-immunized mice. Protection of immunized mice against viral pathogen introduction was evaluated as already described in other examples.

Animals were introduced into the pathogen 15 days after the end of the immunization schedule with 10 ⁶ pfu vvRE by the ip route, and the presence of the virus in the ovaries of mice was assessed 5 days after virus inoculation. 10 shows the results obtained, where the differences observed in virus titers among the groups immunized with different schedules were not statistically significant. In this figure, other negative controls are also included (group 5), corresponding to non-immunized mice.

Example  4: administered by other routes HCV  Assessment of immune response in BALB / c mice after immunization with protein formulation.

Administration of vaccine formulations via other immunization pathways is also one research aspect. Female BALB / c mice are immunized with HCV agents to generate optimal cellular and humoral immune responses by different routes: Group 1: Optimal formulation for humoral responses by subcutaneous (sc) route, Group 2: Optimal formulation for humoral response by intraperitoneal (ip) route, Group 3: Optimal formulation for humoral response by intramuscular (im) route, Group 4: Humoral response by intranasal (in) route For optimal formulation, group 5: sc Optimal formulation for cellular response by route, group 6: i.p optimal formulation for cellular response by route, group 7: i.m. Optimal formulation for cellular response by route, group 8: Optimal formulation for cellular response by route i.n.

Humoral immune responses to HCV structural antigens (cores, El and E2) were studied 15 days after the end of the immunization schedule (0, 1 and 2 weeks). The results obtained show that a specific immune response to the employed antigens occurred despite having statistically significant differences among the antibody levels. These differences are due to the immunization schedule employed, where the occurrence of the antibody response is preferred for some variants due to the kinetics of the immune response. s.c., i.p. And no statistically significant difference was observed when the i.m route was used. However, the results were high when the i.p path was used and low when the i.n path was used for statistically significant differences. The results are shown in FIG.

Lymphoproliferative immune responses in mice were assessed for viral antigens 15 days after the last immunization. Similar to the humoral immune response, the difference in stimulation index among the studied groups depends on the i.n pathway and the amount of antigen used in the general pathway (rest immunization pathway) because of the kinetics of the immune that generated the immune response. Specific lymphoproliferative responses were generated for each HCV antigen. Nevertheless, i.n. The stimulation index obtained by the pathway was statistically significantly lower than other immunization pathways. 12 shows the results obtained. In this assay, another negative control was also included (group 9), corresponding to non-immunized mice.

Protection against viral pathogen introduction in immunized mice was assessed by inoculation 15 days after the end of the immunization schedule with vvRE of 10 ⁶ pfu. Five days after the introduction of the pathogen, the amount of virus was determined in the ovaries of mice immunized and introduced with the pathogen. FIG. 13 shows the results obtained where no difference in other immunization schedules was observed, although a decrease in virus titer of 1 Log in the in pathway was observed. In this experiment, other negative controls corresponding to non-immunized mice are included (group 9).

Example 5: Evaluation of immune response in BALB / c mice after immunization with HCV protein preparations using different adjuvant .

The use of adjuvant has also been studied to enhance the immune response generated by the vaccine formulation. Female BALB / c mice are immunized ip with the vaccine formulation to generate an optimal cellular or optimal humoral immune response as follows: Group 1: Optimal variant for humoral response in aluminum hydroxide (AlOH ₃ ) Group 2: Optimal variant for humoral immune response in aluminum phosphate, Group 3: Optimal variant for humoral immune response in Freund's adjuvant, Group 4: Humoral immunity in oily adjuvant (Montanide ISA 51) Optimal variant for the response, group 5: Optimal variant for the humoral immune response without adjuvant, group 6: optimal variant for the cellular immune response in aluminum hydroxide (AlOH ₃ ), group 7: cells in aluminum phosphate Optimal Variants for Sexual Immune Response, Group 8: Optimal Variants for Cellular Immune Response in Freund's Adjuvant, Group 9: Cellular Immune Responses in Oily Adjuvant (Montanide ISA 51) Optimal variant for cellular immune response without adjuvant: a best elastic element, 10 group. 15 days after the end of the immunization schedule (weeks 0, 1 and 2), the humoral response to HCV structural antigens (Core. E1 and E2) was studied. The results show that in spite of differences in antibody titers in all groups, specific responses were generated for HCV antigens. Levels of antibodies were similar in these groups immunized with vaccine variants in aluminum hydroxide (AlOH ₃ ) and aluminum phosphate. The group immunized with the Freund's adjuvant formulation reached the highest antibody titer followed by the group immunized with the formulation in Montanide ISA 51. Although there was a specific antibody response in the group immunized with the agent without the adjuvant, the antibody titer obtained was the lowest. These results are shown in FIG.

Lymphoproliferative immune responses against HCV viral structural antigens were evaluated in spleen cells from immunized mice 15 days after the end of the immune schedule. The best lymphoid indices were observed in the group immunized with the optimal variant of cell response in Freund's adjuvant and Montanide ISA 51, respectively. Lymphoproliferation indices were similar in the groups immunized with formulations in aluminum hydroxide and aluminum phosphate. The lowest level of cellular immune response was observed when the agent was administered without an adjuvant. These results are shown in FIG. 15, where a negative control (group 11) corresponding to non-immunized mice is included.

Protection against viral pathogen introduction was assessed by inoculation 15 days after the end of the immunization schedule with vvRE of 10 ⁶ pfu. Five days after the introduction of the pathogen, the titer of the virus was assayed in the mouse ovary. Results are shown in FIG. 16 and include additional negative controls (group 11) corresponding to non-immunized mice. In mice immunized with the best variant for the humoral immune response and the best variant for the cellular immune response, both 1.7 and 2.4-Log reductions in virus titer were observed in the Freund's adjuvant, respectively. In these mice immunized with the best variant for the humoral immune response adjuvanted with Montanide ISA 51 and the best variant for the cellular immune response, 1.9 and 2.5-Log reductions in virus titers were observed, respectively. In mice immunized with the preparation of aluminum salts, the decrease in viral titer in the ovaries of the mice was 1.4 Log and 2.1 Log in the case of variants for induction of optimal humoral and optimal cellular immune responses, respectively. Only small decreases in viral titers were observed in animals immunized without an adjuvant.

Example 6: Evaluation of immune response in BALB / c mice after immunization with HCV protein preparations mixed with other viral antigens .

The development or enhancement of immune responses by other viral antigens (hepatitis B surface antigen-HBsAg, hepatitis B core antigen-HBcAg) mixed with HCV agents (HVC agents for generating an optimal cellular immune response) were evaluated in mice. . BALB / c mice were immunized with the i.p pathway: group 1 with HBsAg-HCV vaccine formulation, group 2 with HBcAg-HCV vaccine formulation, group 3 with HCV vaccine formulation, group 4 with HBsAg and group 5 with HbcAg. The immune schedules employed were inoculated at 0, 2 and 4 weeks and the immune response was studied 15 days after the last immunization. Antibody responses were assessed against HCV structural antigens (cores, El and E2) as well as against HBcAg and HbsAg. Occurrence of antigen specific immune responses was observed. Specific antibodies against HBcAg and HbsAg were generated. Although antibody titers against HbcAg in group 2 (HBcAg-HCV vaccine preparation) were somewhat lower than those in the control group (group 5—HBcAg alone), their differences are not statistically significant. Titers to HbsAg were higher in Group 1 (HBsAg-HCV vaccine formulation) compared to Control 4 (HBsAg alone), indicating a tendency to enhance the immune response to this antigen, although the differences observed were not statistically significant. These results are shown in FIG.

Lymphoproliferative immune responses in splenic cells from immunized mice were assessed 15 days after the end of the immunization schedule, against HBcAg and HbsAg as well as against HCV structural virus antigens. As shown in FIG. 18, no difference in stimulation index was found when comparing a mixture of HCV vaccine formulation and viral antigen HBcAg or HbsAg with the corresponding control. In each case, an antigen specific immune response occurred. When stimulus indices from the studied groups were compared, no statistically significant difference was found. In this experiment, additional negative controls (group 6) were included to correspond to non-immunized mice.

Protection against viral pathogen introduction in immunized mice was assessed by inoculation 15 days after the end of the immunization schedule with vvRE of 10 ⁶ pfu. Five days after the introduction of the pathogen, the titer of the virus was assayed in the mouse ovary. 19 shows the results obtained where it was observed that a decrease in virus titer of approximately 2.5 Log was detected only in the group of mice immunized with HCV vaccine formulations that were not mixed with HBV virus antibody (HBsAg or HBcAg). When the viral titers were compared, no statistically significant difference was observed. In the negative control for HCV, no reduction in viral load of the ovary from pathogenic transgenic mice was detected. Additional negative controls (group 6) corresponding to non-immunized mice are included in this experiment.

Example 7: Evaluation of immune response in BALB / c mice after immunization with HCV protein preparation mixed with bacterial antigens .

Bacterial Antigens ( Niceria Mening outer membrane protein from the discharge giti - OMP-) and the HCV vaccine preparation (preparation to generate an HCV optimal immune response) generation of an immune response by the combination or the enhancement was evaluated after the mice immunization. BALB / c mice were immunized with the ip route: group 1 was immunized with OMP-HCV vaccine formulation, group 2 with HCV vaccine formulation and group 3 with OMP adjuvanted with aluminum hydroxide. Mice were immunized at 0, 2 and 4 weeks and immune response was studied 15 days after the end of the immunization schedule. The humoral immune response of older Cuban strain CU 385/83 ceria The formulation of the outer membrane protein from Meningitidis was evaluated as well as for the HCV structural antigens (cores, El and E2). 20 shows that antibody levels generated by the combination of HCV vaccine formulations with OMPs were higher than those generated by OMP alone and HCV vaccine formulations by measuring response to HCV independent antibodies and OMP. These differences are not statistically significant.

Lymphoproliferative responses in splenic cells from immunized mice are Neisseria 15 days after end of immunization schedule It was evaluated for HCV structural antigens as well as for OMP from Meningitidis CU 385/83 Cuban isolate. As shown in FIG. 21, when they were stimulated with HCV structural antigens and OMP, the stimulation index was increased in spleen cells of mice immunized with OMP-HCV vaccine preparation compared to that observed in the control group. This stimulation is antigen specific. In this experiment, additional negative controls (group 4) corresponding to non-immunized mice are included.

Protection after viral pathogen introduction in immunized mice was assessed by inoculation of vvRE of 10 ⁶ pfu 15 days after the end of the immunization schedule. Five days after the pathogen introduction, the titers of the virus were assayed in the ovaries of the pathogen-inducing mice. The obtained result was shown in FIG. There was a 2 Log reduction in virus titers in both groups (HCV vaccine formulation and OMP-HCV vaccine formulation). No decrease in viral titer in the ovary was observed in the control group (mouse immunized with OMP). The same happened with the other negative controls (group 4) included in this experiment, where the mice were not immunized.

The functional activity of antibodies raised against meningitis globules was assessed by analysis of serum-bactericides. In this assay, the ability of the antibody to kill bacteria in vitro in the presence of exogenous supplements was measured. The results shown in Table 3 indicate that the mixture of HCV vaccine formulations with OMP, because bacterial titers were similar in groups 1 and 3, was a Neisseria It does not inhibit or elevate the development of specific-functional antibodies against meningitidis .

Example 8 Evaluation of Immune Responses in BALB / c Mice Following Immunization with HCV Protein Formulation Mixed with Polysaccharides

HCV vaccine preparation (for optimal cellular vaccine preparation to induce an immune response) is a poly sakarayi mixed capsule Drew (P64k carrier protein conjugated to the age ceria The effect on the immune response caused by the encapsulated polysaccharide from Meningitidis serogroup C-) was directed to the BALB / c mice by the ip route: group 1 with the conjugate (MenC-P64k) -HCV vaccine formulation. , Group 2 was assessed after immunization with HCV vaccine formulation and group 3 with conjugate (MenC-P64k). This immunization schedule included vaccinations at weeks 0, 2 and 4 and the immune response was studied 15 days after the final immunization.

Humoral Immune Response in Neisseria Meningitidis serogroup C encapsulated polysaccharides were assessed as well as HCV structural antigens (core, El and E2 proteins).

Figure 23 shows antibody levels generated by the combination of HCV vaccine formulations mixed with conjugates from serogroup C When evaluated for Meningitidis capsular polysaccharide as well as for HCV structural proteins it is shown that they are similar to those obtained independently by both components.

Lymphoproliferative responses in spleen cells of immunized mice were evaluated for HCV structural proteins. As shown in FIG. 24, splenocytes of mice immunized with the vaccine combination MenC-P64k-HCV vaccine preparation and HCV vaccine preparation were stimulated with antigen from the HCV structural region as compared to the control group immunized with the conjugate only. In this experiment, other negative controls were included in group 4, including non-immunized mice.

Protection in immunized mice following viral pathogen introduction was assessed by inoculation of 10 ⁶ pfu vvRE 15 days after the end of the immunization schedule. Five days after the introduction of the pathogen, titration of the virus drop in the ovaries of immunized and pathogen-induced mice was assayed. 25 shows the results, where there is no difference in viral load determined in the ovaries of mice immunized with HCV vaccine formulations with or without the conjugate (MenC-P64k). Compared with the control group, a 2.1-Log decrease in viral titer was observed in both groups. This difference was statistically significant when the virus titers in these groups were compared to the negative control (mouse immunized with conjugate MenC-P64k only). Additional negative controls (group 4) corresponding to unimmunized mice were included in this experiment.

Neisseria When the functional activity of antibodies raised against Meningitidis serogroup C was in the presence of exogenous supplements, the ability of the antibodies to kill bacteria was assessed by analysis of fungicides measured in vitro. In this sense, the results shown in Table 4 HCV vaccine - MenC-P64K age the mixture of the conjugate produced by the conjugate MenC-P64K ceria It does not affect the development of specific and functional antibodies against meningitidis .

Example 9: Evaluation of after immunization with HCV protein preparations mixed with a plasmid encoding the viral antigen BALB / c mice immune response.

Code for the surface antigen (pAEC-M7-HBsAg) and core antigen (pAEC-M7-HBcAg) of hepatitis B virus in the development of an immune response when mixed with HCV vaccine preparation (optimal variant for cellular immune response). Effects of DNA immunization plasmids on BALB / c mice: group 1 as pAEC-M7, group 2 as pAEC-M7-HCV vaccine preparation, group 3 as pAEC-M7-HbsAg, group 4 as pAEC-M7- HBsAg-HCV vaccine formulations were studied after intramuscular immunization with group 5 with pAEC-M7-HbcAg, group 6 with pAEC-M7-HBcAg-HCV vaccine formulation and group 7 with HCV vaccine formulation. Animals were immunized at 0, 2, 4 and 6 weeks. Humoral immune responses to HCV structural antigens (cores, El and E2) as well as to hepatitis B virus surface (HBsAg) and core (HBcAg) antigens were evaluated. The results obtained are shown in FIG. 26, where the occurrence of specific responses to each of the analyzed antigens can be observed. In general, when plasmids were mixed into HCV vaccine preparations, antibody titers against HCV structural antigens were higher than those generated by HCV vaccine preparations alone. Although this was some tendency, the observed differences were not statistically significant. When animals were immunized with plasmids encoding HBsAg and HbcAg alone or in combination with HCV vaccine formulations, antibody titers against these antigens were similar.

Lymphoproliferative responses in splenic cells from immunized mice were assessed 15 days after the end of the immunization schedule for HCB structural antigens as well as for HBsAg and HbcAg. As shown in FIG. 27, no statistical difference in stimulation index was observed when the HCV vaccine formulation mixed with the plasmid encoding hepatitis B antigen (HBsAg or HBcAg) was compared with the corresponding control. In this experiment, additional negative controls (group 8) were included, including non-immunized mice. In each case, antibody-specific responses were observed. When the groups studied were compared, the difference detected in the stimulation indices was not statistically significant. At the same time, no statistically significant differences were observed between groups of mice immunized with HCV vaccine preparations with or without plasmid mixed when HCV construct antigen was added to splenic cells.

Protection after viral pathogen introduction in immunized mice was assessed by inoculation of vvRE of 10 ⁶ pfu 15 days after the end of the immunization schedule. Five days after the introduction of the pathogen, virus dropping was assayed in the ovaries of the pathogen-induced mice. FIG. 28 shows the results obtained, where there was a decrease of approximately 2 Log in virus titers only in groups immunized with HCV vaccine formulations with or without plasmids mixed. There was no statistically significant difference when the titers of viruses were compared between these groups. In the HCV negative control, there was no reduction of viral load in the ovaries of immunized mice. In this experiment, additional negative controls (group 8) corresponding to non-immunized mice were included.

Example 10: Evaluation of immune response in BALB / c mice after further immunization with HCV protein preparations studied with initial immunization with DNA formulation encoding for HCV structural regions .

Combination of initial immunity with DNA and additional immunity with HCV vaccine formulation (for optimal cellular immune response) was studied in BALB / c mice. Immunization schedules are shown in Table 5.

BALB / c mice were immune by the im route in a DNA plasmid formulation (Duens-Carrera, et al. (2002). Enhancement of the immune response generated against the hepatitis C virus envelope proteins after DNA vaccination with polyprotein-encoding plasmids. Biotechnol Appl Biochem , 35, 205-212) Additional doses were administered by the im route.

Humoral immune responses were assessed for HCV structural antigens (core, El and E2). The obtained result was shown in FIG. No evidence of enhancement of antibody response to HCV core antigen was found. The antibody levels detected in these groups immunized with protein variants are higher than for the DNA immunized group, although not statistically significant.

The humoral immune response to El protein was elevated when further administered with HCV vaccine formulation. In this case, higher antibody levels were detected in the group of mice immunized with protein variants compared to the group immunized with DNA. Before and after further dosing, the difference observed in antibody levels against E1 was not statistically significant.

Antibody levels detected against E2 protein using this immunization regimen were higher in animals immunized with protein formulation compared to antibody responses obtained in mice immunized with DNA. The antibody level detected against E2 protein did not change after further doses.

Lymphoproliferative responses in spleen cells of immunized mice were assessed for rescue HCV virus antigens 15 days after the last immunization. As shown in FIG. 30, although the lymphoproliferation index was increased in spleen cells from mice added with protein formulation, the difference is not statistically significant. Similarly, lymphoproliferative responses to HCV structural antigens of mice immunized with DNA and added to proteins were markedly increased for El protein. Differences between groups are not statistically significant.

Protection against viral pathogen introduction in immunized mice was assessed by inoculation of vvRE of 10 ⁶ pfu 15 days after the last immunization. Five days after the pathogen introduction, the titer of the virus was determined in the ovaries of immunized and pathogen-induced mice. FIG. 31 shows the results obtained, where approximately a 2 Log reduction in viral load was detected in animals immunized with the optimal cell vaccine formulation. Mice immunized with DNA showed a 1.2 Log decrease in viral titer. After further dose with vaccine formulation for optimal cellular immune response, a 6.6 Log reduction in virus titer was observed in animals initially immunized with the formulation for optimal cellular response. A 1.8 Log reduction in viral titer was observed in mice initially immunized with DNA and further immunized with HCV vaccine preparation. In other groups, no decrease in viral load was found in the ovaries of mice.

Example 11: Evaluation of immune response in BALB / c mice after immunization with HCV protein preparations taking into account other routes of preparations .

Induction of immune responses against recombinant HCV structural proteins has been studied after preparation of immunogens by other forms. The following variants were studied: (1) three antigens were mixed and then adjuvanted with aluminum hydroxide, (2) each antigen was first adjuvanted separately with aluminum hydroxide gel and later mixed to make up the final formulation. BALB / c mice were immunized in both formulations. Vaccine formulations were used to induce optimal cellular responses in BALB / c mice.

Established immunization schedules are 0, 2 and 4 weeks. The immune response was studied 15 days after the immunization schedule was completed.

Antibody responses were assessed for antigens in which HCV vaccine preparations (cores, El and E2) were present. 32 shows similar antibody levels generated for each antigen; Differences between the groups studied indicate that they were not statistically significant.

Lymphoproliferative responses in splenic cells of immunized mice were evaluated for HCV structural antigens. As shown in FIG. 33, the method of preparing the HCV immunogen did not affect the antibody-specific lymphoproliferative response. The difference between the groups studied was not statistically significant. Other controls are included in this experiment (group 3) and correspond to non-immunized mice.

Protection of immunized mice against viral pathogen introduction was assessed by vvRE inoculation of 10 ⁶ pfu 15 days after the last immunization. Five days after pathogen introduction, viral titers were determined in the ovaries of immunized and pathogen-induced mice. The amount of virus detected in the ovary of immunized mice is expressed as Log of virus titers, indicating that the differences between the groups studied were not statistically significant. These results are shown in FIG. Other controls are included in this experiment (group 3) and correspond to non-immunized mice.

Claims (13)

A vaccine composition for generating a protective cellular immune response against infection with hepatitis C virus, characterized by a combination of hepatitis C virus core, E1 and E2 structural antigens with a weight ratio of 1: 160: 160. The vaccine composition of claim 1, wherein the vaccine composition is administered on a different immunization schedule. The vaccine composition of claim 1, wherein the vaccine composition is administered after initial immunization with a DNA formulation encoding for an antigen of an HCV structural region. A combined vaccine composition for viral or bacterial diseases wherein a mixture of hepatitis C virus structural antigens as an active ingredient is combined with a polysaccharide (conjugated or not) or a viral or bacterial antigen according to claim 1. The vaccine composition of claim 1 for inducing immunity to hepatitis C virus in a patient with chronic hepatitis C, cirrhosis, and liver cancer. delete delete delete delete delete delete delete delete

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